This grant focuses on the basic cell biological process of membrane trafficking through the secretory and endocytic pathways in mammalian cells. Secretion and endocytosis play fundamental biological roles at the levels of both single cells and multi-cellular organisms to control a wide array of physiological processes including nutrient uptake, secretion of hormones and digestive enzymes, and defense against foreign pathogens. Secretion and endocytosis involve the transport of cargo between intracellular organelles of these pathways, e.g., between the endoplasmic reticulum (ER) and Golgi complex. Defects in the transport of proteins through these pathways are associated with a host of diseases including cancer, cystic fibrosis, and atherosclerosis. In mammalian cells, membrane tubules (60-80 nm in diameter and to many microns in length) emanate from various organelles of the secretory and endocytic pathways, and these tubules have been implicated in many intracellular trafficking steps, including retrograde trafficking from the Golgi complex to the ER and recycling from receptors from endosomes to the cell surface. Recent evidence from my laboratory shows that cytoplasmic Ca2+independent phospholipase A2 (PLA2) and lysophospholipid acyltransferase (LPAT) enzymes, with opposing enzymatic activities, may work in concert to mediate tubule formation by directly altering membrane phospholipid composition and consequently membrane shape. The long-term objectives of this grant are to elucidate the roles of membrane tubules in intracellular trafficking, and to determine the biological functions of specific PLA2 and LPAT enzymes with respect to secretion and endocytosis in mammalian cells. The goals of this work are encompassed in two large Specific Aims: 1) identify and characterize the PLA2 enzymes and accessory proteins involved in stimulating tubule formation; 2) identify and characterize the LPATs involved in mediating tubule formation and/or coated vesicle fission. These goals will be achieved through a combination of molecular, cellular, and genetic approaches. The in vivo roles of candidate PLA2 and LPAT enzymes in secretory and endocytic trafficking will be investigated by conducting over-expression and siRNA knock-down experiments. These experiments will focus on Golgi-to-ER retrograde trafficking, export from endocytic compartments, and vesicle fission from membrane-bound organelles. In vitro assays that reconstitute tubule or vesicle formation will also be conducted to elucidate the mechanisms underlying the effects of PLA2 activity on membrane shape. These studies will reveal novel biological roles for PLA2 and LPAT enzymes in mediating intracellular trafficking events in the secretory and endocytic pathways. [unreadable] [unreadable]